Cells are often cultured in reactors for generation of useful cell products, such as anti-bodies. In order to maintain a cell culture, oxygen and other nutrients generally must be supplied to the cells. Cell cultures are usually maintained in reactors by perfusion, wherein a cell culture medium, including oxygen and other nutrients, is directed through the cell-culture reactor.
However, cell-culture reactors which are currently in use often suffer from other types of problems. They typically can support only small cell loadings per unit of reactor volume. Also, they can operate only within a small window of flow or agitation rates and they usually require other cell-retention devices (i.e. filters) in order to be able to operate in a perfusion mode. Their capacity in sparingly soluble nutrients, such as oxygen, is small, and as a result, scale-up problems are not easy to solve. For the case of beds of substrates, the height of beds is limited by the solubility of oxygen in the medium directed through the cell culture.
One specific problem is that use of porous particles as substrates for perfused cell cultures has been limited. The supply of nutrients, and, in particular, of oxygen, is thereby limited due to small rates at which such nutrients are transported by diffusion from the bulk to the interior of the particle where cells typically reside. As a result, the total number of cells that can be supported by diffusive nutrient supply is small and their production capability usually low. Further, the density of cells within the pores has been difficult to control because the rate of diffusion into the pores generally is not significantly affected by the rate of flow of medium through the interstitial passages. Non-porous microcarriers have no such transport limitations, however, their applicability is seriously impaired by high sensitivity to shear, low cell densities and low bioreactor productivities.
Further, suitable media for culturing cells typically exhibit a limited solubility of oxygen, thereby causing medium directed through a cell culture reactor to be depleted of oxygen prior to depletion of other nutrients. Cell-culture reactor systems, therefore, generally have included recycling of the medium through a remote reservoir in order to replenish the oxygen content of the medium. Medium is typically recycled in order to obtain a concentration of product in the medium which can be processed from the medium cost-effectively, and also to increase the amount of product per unit volume of spent medium.
However, cell productivity is limited in recycle-type reactors because medium which is recycled through the remote reservoir often cannot be treated to remove cell waste-products which exhibit toxicity to the cell culture. The productivity per cell in the cell culture of desired products is thereby diminished as waste products accumulate in the medium which is recirculated through the cell culture and the cell culture reactor.
There have been many attempts to increase the concentration of oxygen in medium directed through cell culture reactors. However, most methods have suffered from various types of problems. For example, increasing the rate of oxygen supplied to a cell culture reactor by increasing the rate of medium flow through the cell culture reactor is limited by the high sensitivity of cells to shear forces resulting from the increased rate of medium flow. Sparging of oxygen directly into a cell culture reactor is usually unacceptable, especially for mammalian cell cultures, because of the susceptibility of such cultures to detrimental proximate hydrodynamic forces and excessive foaming of the medium as a consequence of sparging. In another example, perfluorocarbons have been introduced to a medium in order to increase the solubility of oxygen in the medium. However, perfluorocarbons must be separated from the medium prior to recovery of cell products, thereby introducing an additional processing step which can reduce productivity and add expense to the operation of cell-culture reactors.
Entrapment of cells within pores of a macroporous support in order to protect cells from damaging shearing forces allows faster rates of medium flow through a cell culture reactor. However, in the absence of fluid flow through the pores of the support, transport of oxygen and other nutrients to cells can be limited by the process of diffusion. Also, although enabling a larger cell culture reactor, the medium must still be recycled in order to attain better medium utilization and cost-effective concentrations of cell product, thereby resulting in introduction of medium to the cell culture which contains a significant amount of cell waste-products.
A need exists, therefore, for a new method and apparatus for culturing cells which overcome or minimize the aforementioned problems.